I propose to study a mechanism for regulation of reversible bacterial cell differentiation which L. monocytogenes exhibits both in vitro and in colonized animals. The studied differentiation step involves phase-variation in the completion of L. monocytogenes septation during cell-division, which manifests as smooth (S) or rough (R) colony morphologies. I will identify the lesion in a transposon mutagenized, R-phase-locked L. monocytogenes strain. The effect that expression of this disrupted locus (termed efs) has on L. monocytogenes morphology will be evaluated by studying the morphological consequences of efs deletion and overexpression. Subsequently, mutants locked in S-phase will be engineered by altering expression of the efs locus, or isolated in screens of mutagenized L. monocytogenes. These phase-locked strains will be used to study the effects of phase-variation on the ability of L. monocytogenes to compete for colonization of the gut and internal organs of orally and intravenously-infected mice. The phase-locked L. monocytogenes strains will also be used to evaluate the effects of phase-variation on development of host T cell immune responses to this pathogen. For this experiment, CTL responses to a subset of previously-defined peptide epitopes from bacterial proteins will be quantitated. The expression of these bacterial proteins is known to differ in S-phase and R-phase L. monocytogenes. The studies in this proposal should provide insight into a potentially novel mechanism for regulation of bacterial cell differentiation and will begin to address how this mechanism affects virulence and immunogenicity of L. monocytogenes in a murine host.